HIV-associated nephropathy (HIVAN) is a renal disease almost exclusively seen in people of African ancestry. More than 2 million HIV-infected children living in sub-Saharan Africa are at high risk of developing HIVAN, if they do not receiving appropriate anti-retroviral therapy (ART). HIVAN is characterized by the collapse of glomerular capillaries and micro-cystic transformation of renal tubules leading to rapid chronic renal failure or death. These changes are caused by the infection of podocytes and renal tubular epithelial cells (RTEc), yet the mechanism is unclear. Two genetic risk variants in the human APOL1 gene (G1/G2) were identified as major risks factors for developing HIVAN in people of African ancestry. Nonetheless, other endogenous factors are needed as well, since people of African ancestry who do not carrying the ApoL-1 risk variants, or HIV- transgenic (Tg) mice, also develop HIVAN. Our program is in a unique position to identify these factors, since we have been exploring this topic in children for the last 20 years funded by the current R0-1, and have collected a large number of samples and tissues during this time. In addition, during the last cycle of the application, we have (1) identified a new role for Tumor Necrosis Factor-alpha (TNF-a) facilitating the establishment of a productive infection of podocytes cultured from children with HIVAN; and found that (2) HIV-Tat is preferentially recruited to lipid rafts (LRs) in podocytes cultured from children with HIVAN, enhancing the signaling of heparin binding growth factors (HBGF) and activating the expression of HIV-genes. Based on these data, we hypothesize that trans-membrane TNF-a, modulates the infection and injury of renal epithelial cells by interacting with heparan sulfate proteoglycans, lipid rafts (LRs), and globotriaosylceramide (Gb3). A second corollary of this hypothesis is that the recruitment of HIV-Tat to LRs, in synergy with other HBGF, precipitates the collapse of glomerular capillaries. Finally, we hypothesize that HBGF release in the urine of children with HIVAN can become reliable biomarkers to diagnose HIVAN and to follow the clinical outcome of these patients in combination with other non-invasive tests. To test these hypotheses, in aim 1, we will define how trans- membrane TNF-a modulates the infection of podocytes and renal tubular epithelial cells (RTEc) cultured from children with HIVAN by interacting with LRs, and Gb3. In aim 2, we will determine how HIV-1 and HIV-Tat, precipitate the collapse of glomerular capillaries by interacting with the Von Hippel-Lindau protein (pVHL) and HIF-2a-inducible HBGF in cultured podocytes from children with HIVAN and new transgenic mice. In aim 3, we will determine the clinical value of a new panel of urinary biomarkers, a podocyte permeability assay, podocyturia, and the APOL-1 genotype, to diagnose HIVAN in children and young adults, and to follow their clinical outcome. In summary, these studies will generate fundamental new knowledge to improve our understanding of the pathogenesis HIVAN, and identify new urinary biomarkers and other non-invasive tests that will be used to establish the diagnosis of HIVAN in children without doing a renal biopsy.